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目的克隆人纤溶酶原激活物抑制物1(PAI1)基因,制备和鉴定针对人 PAI1的单克隆抗体,以检测 PAI1在乳腺癌中的表达。方法利用逆转录(RT)-PCR 方法从人乳腺癌细胞株MDA231总 RNA 中扩增克隆 PAI1基因,构建其原核表达质粒,并表达 MS2-PAI1融合蛋白。以纯化的融合蛋白免疫 BALB/C 小鼠,常规细胞融合和选择性培养,亚克隆筛选出单克隆细胞株,纯化单克隆抗体。利用蛋白印迹(Wb)、免疫组织化学法检测分析 PAI1在乳腺癌中的表达水平。结果克隆了1209 bp 的 PAI1基因全长片段。经 ELISA 双筛,获得2株能稳定分泌抗 PAI1单克隆抗体的杂交瘤细胞株,其分泌抗体亚类均为 IgG1,且均能特异识别 PAI1,并与其他蛋白无交叉反应。Wb 结果显示,该抗 PAI1单抗与 MS2-PAI1融合蛋白以及细胞中的天然蛋白均能结合。免疫组织化学染色结果显示,该单抗与人乳腺癌细胞株 MDA231和乳腺癌组织结合后,呈阳性反应。结论克隆了人 PAI1基因,通过原核表达产物制备并纯化了针对 PAI1的单克隆抗体。初步检测 PAI1能在乳腺癌细胞和乳腺癌组织中表达,这为深入研究 PAI1基因功能和检测临床肿瘤组织及体液中的 PAI1奠定了可靠基础。
Objective To clone the human plasminogen activator inhibitor 1 (PAI1) gene and prepare and identify a monoclonal antibody against human PAI1 to detect the expression of PAI1 in breast cancer. Methods PAI1 gene was amplified from total RNA of human breast cancer cell line MDA231 by reverse transcription (RT) -PCR method. The prokaryotic expression plasmid was constructed and the fusion protein of MS2-PAI1 was expressed. The purified fusion protein was used to immunize BALB / C mice. Conventional cell fusion and selective culture were performed. The monoclonal cell lines were subcloned and monoclonal antibodies were purified. The expression level of PAI1 in breast cancer was analyzed by immunohistochemistry with Western blotting (Wb). Results The PAI1 full-length fragment of 1209 bp was cloned. Two hybridoma cell lines secreting anti-PAI1 monoclonal antibodies were obtained by double-ELISA screening. The secreted antibody subtypes were both IgG1, and all of them could specifically recognize PAI1 and did not cross-react with other proteins. The Wb results show that the anti-PAIl mAb binds to both the MS2-PAI1 fusion protein and the native protein in the cell. Immunohistochemical staining results showed that the monoclonal antibody showed positive reaction with human breast cancer cell line MDA231 and breast cancer tissue. Conclusion The human PAI1 gene was cloned and the monoclonal antibody against PAI1 was prepared and purified by prokaryotic expression. The preliminary detection of PAI1 expression in breast cancer cells and breast cancer tissue, which in-depth study of PAI1 gene function and detection of clinical tumor tissue and body fluid PAI1 laid a solid foundation.